E. coli o157:h7 specific assay

ABSTRACT

Disclosed are assays, methods and kits for the specific detection of  E. coli  O157:H7 and not  E. coli  O55:H7 from complex food matrices, water, a beverage sample, a fermentation broth, a forensic sample, an environmental sample (e.g., soil, dirt, garbage, sewage, air, or water), including food processing and manufacturing surfaces, or a biological sample.

CROSS-REFERENCE(S) TO RELATED APPLICATION(S)

This application claims the benefit under 35 U.S.C. §119(e) of U.S.Provisional Application No. 61/178,931, filed May 15, 2009, the contentsof which is incorporated herein by reference.

FIELD OF THE INVENTION

The present teachings relate to assays and methods for the specificdetection and differentiation of pathogenic organisms.

BACKGROUND

Identification of bacterial contamination in food often occurssubsequent to an outbreak of a foodborne illness. The bacteriumEscherichia coli is frequently identified as the food contaminant ofmany foodborne illnesses. The serotype known as E. coli O157:H7 causesenterohemorrhagic colitis and possibly kidney failure. It often resultsin hospitalization of the infected patient and can be particularlylethal in young children and the elderly. O157:H7 is most oftenassociated with outbreaks of foodborne illness in the United States andelsewhere in the world.

Detection of pathogenic E. coli, particularly serotypes causative ofhemorrhagic colitis has become a public health priority. O157:H7 isfrequently isolated from cattle, including healthy animals and has alsobeen associated with illness in contaminated produce. The presence ofO157:H7 in a food product released to consumers is considered asevidence of adulteration of the product. Regulations by the UnitedStates government require meat processors to screen for the presence ofO157:H7 in their finished products and more stringent guidelines arebeing considered in a number of states for the identification of O157:H7in other commodities and food stuffs. An assay for the rapid, sensitiveand specific detection of infectious pathogens is extremely importantfrom both a public health and economic perspective.

Many strains of genetically similar E. coli exist that vary dramaticallyin their pathogenicity. Genomic comparisons are revealing theconsequences of genetic changes often underlie the emergence of newpathogenic bacteria. E. coli O157:H7 has been determined to have evolvedstepwise from the O55:H7 which is associated with infantile diarrhea.These two serotypes are more closely related at the nucleotide levelwhile divergence was markedly different at the gene level. Likewise,other pathogenic serotypes have been shown to be less divergent at thenucleotide level making identification of pathogenic strains difficult.

An assay utilizing molecular methods such as sequence specificamplification and detection offer significant improvements in speed,sensitivity and specificity over traditional microbiological methods.Design and development of a molecular detection assay that requires theidentification of a target sequence that is present in all organisms tobe detected and absent or divergent in organisms not to be detected isan unmet need for the definitive detection of the O157:H7 serotype of E.coli.

SUMMARY

In accordance with the embodiments, there is disclosed a method ofdetecting the presence of E. coli O157:H7 in a sample, comprising:detecting the presence of SEQ ID NO:111 or complement thereof; anddetecting the presence of a sequence selected from SEQ ID NO:90-110 andcomplements thereof; wherein detection of SEQ ID NO:111 and detection ofa sequence selected from SEQ ID NO:90-110 confirms the presence of E.coli O157:H7 in a sample and not E. coli O55:H7. The detection is by anucleic acid amplification reaction, the amplification reaction is anend-point determination, the amplification reaction is quantitative, thequantification is a real-time PCR, the real-time PCR is a SYBR® GreenAssay or the real-time PCR is a TaqMan® Assay.

In another embodiment, disclosed is an assay for the detection of E.coli

O157:H7 in a sample comprising a) hybridizing a first pair of PCRprimers selected from the group consisting of: SEQ ID NO:1-2, SEQ IDNO:1 and SEQ ID NO:4, SEQ ID NO:1 and SEQ ID NO:5, and SEQ ID NO:1 andSEQ ID NO:6 and complements thereof to at least a first targetpolynucleotide sequence; b) hybridizing a second pair of PCR primersselected from SEQ ID NO:7-8, SEQ ID NO:10-11, SEQ ID NO:13-14, SEQ IDNO:16-17, SEQ ID NO:19-20, SEQ ID NO:22-23, SEQ ID NO:25-26, SEQ IDNO:28-29, SEQ ID NO:31-32, SEQ ID NO:34-35, SEQ ID NO:37-38, SEQ IDNO:40-41, SEQ ID NO:43-44, SEQ ID NO:46-47, SEQ ID NO:49-50, SEQ IDNO:52-53, SEQ ID NO:55-56, SEQ ID NO:59 and SEQ ID NO:56, SEQ IDNO:61-62, SEQ ID NO:64-65, SEQ ID NO:67-68, SEQ ID NO:70-71, SEQ IDNO:73-74, SEQ ID NO:76-77, SEQ ID NO:79-80, SEQ ID NO:82-83, SEQ IDNO:85-86, and SEQ ID NO:88-89 and complements thereof to at least asecond target polynucleotide sequence; c) amplifying said at least firstand said at least second target polynucleotide sequences; and d)detecting said at least first and said at least second amplified targetpolynucleotide sequence products; wherein the detection of the at leastfirst amplified target polynucleotide sequence product and the detectionof the at least second amplified target polynucleotide sequence productis indicative of the presence of E. coli O157:H7 in the sample and notE. coli O55:H7.

In further embodiments, the assay further has a first probe of SEQ IDNO:3 and a second probe selected from SEQ ID NO:9, SEQ ID NO:12, SEQ IDNO:15, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:27 SEQ IDNO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:42, SEQ IDNO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:57, SEQ IDNO:58, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:69, SEQ IDNO:72, SEQ ID NO:75, SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:84, SEQ IDNO:87, and SEQ ID NO:90, the first probe further comprises a first labeland said second probe further comprises a second label, wherein bothlabels are selected from a dye, a radioactive isotope, achemiluminescent label, and an enzyme, the dye comprises a fluoresceindye, a rhodamine dye, or a cyanine dye, the dye is a fluorescein dye andfirst probe is labeled with FAM™ dye and said second probe is labeledwith VIC® dye. The assay further has preparing the sample for PCRamplification prior to hybridizing, for example, but not limited to (1)bacterial enrichment, (2) separation of bacterial cells from the sample,(3) cell lysis, and (4) total DNA extraction, the sample can be for or awater sample, an environmental sample, and so on and the food samplecomprises a selectively enriched food matrix. The assay can be bypolymerase chain reaction, wherein hybridizing and amplifying of saidfirst pair of polynucleotide primers occurs in a first vessel and saidhybridizing and amplifying of said second pair of polynucleotide primersoccurs in a second vessel, or hybridizing and amplifying of said firstpair of polynucleotide primers and said hybridizing and amplifying ofsaid second pair of polynucleotide primers occurs in a single vessel,the detection is a real-time assay, the real-time assay is a SYBR® Greendye assay or a TaqMan® assay.

In still another embodiment, the invention teaches a assay for thedetection of E. coli O157:H7 in a sample comprising: a) hybridizing afirst pair of PCR primers to a first target polynucleotide sequencewithin SEQ ID NO:111; b) hybridizing a second pair of PCR primers to asecond target polynucleotide sequence within a sequence selected fromSEQ ID NO:91-110; c) amplifying said at least first and said at leastsecond target polynucleotide sequences; and d) detecting said at leastfirst and said at least second amplified target polynucleotide sequenceproducts; wherein the detection of the at least first amplified targetpolynucleotide sequence product and the detection of the at least secondamplified target polynucleotide sequence product is indicative of thepresence of E. coli O157:H7 in the sample and not E. coli O55:H7. Theassay can further have a first probe of SEQ ID NO:3 and a second probeselected from SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:18, SEQID NO:21, SEQ ID NO:24, SEQ ID NO:27 SEQ ID NO:30, SEQ ID NO:33, SEQ IDNO:36, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ IDNO:51, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:60, SEQ IDNO:63, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:72, SEQ ID NO:75, SEQ IDNO:78, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:87, and SEQ ID NO:90, thefirst probe further comprises a first label and said second probefurther comprises a second label, wherein both labels are selected froma dye, a radioactive isotope, a chemiluminescent label, and an enzyme,the dye comprises a fluorescein dye, a rhodamine dye, or a cyanine dye,the dye is a fluorescein dye and first probe is labeled with FAM™ dyeand said second probe is labeled with VIC® dye. The assay further haspreparing the sample for PCR amplification prior to hybridizing, forexample, but not limited to (1) bacterial enrichment, (2) separation ofbacterial cells from the sample, (3) cell lysis, and (4) total DNAextraction, the sample can be for or a water sample, an environmentalsample, and so on and the food sample comprises a selectively enrichedfood matrix. The assay can be by polymerase chain reaction, whereinhybridizing and amplifying of said first pair of polynucleotide primersoccurs in a first vessel and said hybridizing and amplifying of saidsecond pair of polynucleotide primers occurs in a second vessel, orhybridizing and amplifying of said first pair of polynucleotide primersand said hybridizing and amplifying of said second pair ofpolynucleotide primers occurs in a single vessel, the detection is areal-time assay, the real-time assay is a SYBR® Green dye assay or aTaqMan® assay.

In one embodiment, disclosed is a method for specifically detecting E.coli O157:H7, comprising: hybridizing at least a first pair ofpolynucleotide primers to at least a first target polynucleotidesequence, hybridizing at least a second pair of polynucleotide primersto at least a second target polynucleotide sequence, amplifying said atleast first and said at least second target polynucleotide sequences,and

detecting said at least first and said at least second amplified targetpolynucleotide sequence products, wherein the detection of the at leastfirst amplified target polynucleotide sequence product and the detectionof the at least second amplified target polynucleotide sequence productis indicative of the presence of E. coli O157:H7 in a sample and not E.coli O55:H7. The method further has preparing the sample for PCRamplification prior to hybridizing, for example, but not limited to (1)bacterial enrichment, (2) separation of bacterial cells from the sample,(3) cell lysis, and (4) total DNA extraction, the sample can be for or awater sample, an environmental sample, and so on and the food samplecomprises a selectively enriched food matrix. The method can be bypolymerase chain reaction, having at least a first probe and at least asecond probe, said first probe further comprises a first label and saidsecond probe further comprises a second label, both labels are selectedfrom a dye, a radioactive isotope, a chemiluminescent label, and anenzyme, the dye comprises a fluorescein dye, a rhodamine dye, or acyanine dye, the first probe is labeled with FAM™ dye and said secondprobe is labeled with VIC® dye, wherein hybridizing and amplifying ofsaid first pair of polynucleotide primers occurs in a first vessel andsaid hybridizing and amplifying of said second pair of polynucleotideprimers occurs in a second vessel, or hybridizing and amplifying of saidfirst pair of polynucleotide primers and said hybridizing and amplifyingof said second pair of polynucleotide primers occurs in a single vessel,the detection is a real-time assay, the real-time assay is a SYBR® Greendye assay or a TaqMan® assay. The method includes a first primer pair isselected from SEQ ID NO:1-2, SEQ ID NO:1 and SEQ ID NO:4, SEQ ID NO:1and SEQ ID NO:5, and SEQ ID NO:1 and SEQ ID NO:6 and said second primerpair is selected from SEQ ID NO:7-8, SEQ ID NO:10-11, SEQ ID NO:13-14,SEQ ID NO:16-17, SEQ ID NO:19-20, SEQ ID NO:22-23, SEQ ID NO:25-26, SEQID NO:28-29, SEQ ID NO:31-32, SEQ ID NO:34-35, SEQ ID NO:37-38, SEQ IDNO:40-41, SEQ ID NO:43-44, SEQ ID NO:46-47, SEQ ID NO:49-50, SEQ IDNO:52-53, SEQ ID NO:55-56, SEQ ID NO:59 and SEQ ID NO:56, SEQ IDNO:61-62, SEQ ID NO:64-65, SEQ ID NO:67-68, SEQ ID NO:70-71, SEQ IDNO:73-74, SEQ ID NO:76-77, SEQ ID NO:79-80, SEQ ID NO:82-83, SEQ IDNO:85-86, and SEQ ID NO:88-89, and said first probe is SEQ ID NO:3 foruse for said first primer pair and said second probe is selectedaccording to FIG. 3 for use with said second primer pair from SEQ IDNO:9, SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21, SEQ IDNO:24, SEQ ID NO:27 SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ IDNO:39, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ IDNO:54, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:63, SEQ IDNO:66, SEQ ID NO:69, SEQ ID NO:72, SEQ ID NO:75, SEQ ID NO:78, SEQ IDNO:81, SEQ ID NO:84, SEQ ID NO:87, and SEQ ID NO:90, wherein said firsttarget polynucleotide sequence is within SEQ ID NO:111 and said secondtarget polynucleotide sequence is within the group selected from SEQ IDNO:91-110.

In another embodiment, disclosed is a polynucleotide sequence or itscomplement for the detection of E. coli O157:H7 and not E. coli O55:H7identical to at least 16 contiguous polynucleotides from a firstsequence selected from the group consisting of SEQ ID NO:90-110 and asecond sequence of SEQ ID NO:111, the contiguous polynucleotide is aprimer or a probe, the has a label selected from a dye, a radioactiveisotope, a chemiluminescent label, and an enzyme, the dye comprises afluorescein dye, a rhodamine dye, or a cyanine dye, including but notlimited to FAM™ dye and VIC® dye, as exemplary dyes.

In another embodiment, disclosed is a kit for the detection of E. coliO157:H7 in a sample comprising a first pair of PCR primers selected fromthe group consisting of: SEQ ID NO:1-2, SEQ ID NO:1 and SEQ ID NO:4, SEQID NO:1 and SEQ ID NO:5, and SEQ ID NO:1 and SEQ ID NO:6; b) a secondpair of PCR primers selected from SEQ ID NO:7-8, SEQ ID NO:10-11, SEQ IDNO:13-14, SEQ ID NO:16-17, SEQ ID NO:19-20, SEQ ID NO:22-23, SEQ IDNO:25-26, SEQ ID NO:28-29, SEQ ID NO:31-32, SEQ ID NO:34-35, SEQ IDNO:37-38, SEQ ID NO:40-41, SEQ ID NO:43-44, SEQ ID NO:46-47, SEQ IDNO:49-50, SEQ ID NO:52-53, SEQ ID NO:55-56, SEQ ID NO:59 and SEQ IDNO:56, SEQ ID NO:61-62, SEQ ID NO:64-65, SEQ ID NO:67-68, SEQ IDNO:70-71, SEQ ID NO:73-74, SEQ ID NO:76-77, SEQ ID NO:79-80, SEQ IDNO:82-83, SEQ ID NO:85-86, and SEQ ID NO:88-89; c) a polymerase; andoptionally a first probe of SEQ ID NO:3 for use with selected said firstpair of PCR primers and a second probe according to FIG. 3 for use withselected said second pair of PCR primers selected from SEQ ID NO:9, SEQID NO:12, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:24, SEQ IDNO:27 SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:39, SEQ IDNO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:54, SEQ IDNO:57, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:66, SEQ IDNO:69, SEQ ID NO:72, SEQ ID NO:75, SEQ ID NO:78, SEQ ID NO:81, SEQ IDNO:84, SEQ ID NO:87, and SEQ ID NO:90. The first probe further comprisesa first label and said second probe further comprises a second label,both labels are selected from a dye, a radioactive isotope, achemiluminescent label, and an enzyme, the dye comprises a fluoresceindye, a rhodamine dye, or a cyanine dye, and the first probe is labeledwith FAM™ dye and said second probe is labeled with VIC® dye.

In the following description, certain aspects and embodiments willbecome evident. It should be understood that a given embodiment need nothave all aspects and features described herein. It should be understoodthat these aspects and embodiments are merely exemplary and explanatoryand are not restrictive of the invention.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention, as claimed.

The accompanying figures, which are incorporated in and constitute apart of this specification, illustrate several exemplary embodiments ofthe disclosure and together with the description, serve to explaincertain teachings.

There still exists a need for improved assays and methods for detectingand differentiating pathogenic organisms from non-pathogenic organismswhich can be complicated by globally regional differences as well as bythe need for improved sensitivity and specificity of detection.

These and other features of the present teachings are set forth herein.

DESCRIPTION OF THE DRAWINGS

The skilled artisan will understand that the drawings described beloware for illustration purposes only. The drawings are not intended tolimit the scope of the present teachings in any way.

FIG. 1 illustrates primer and probe sets useful in detecting SEQ IDNO:111.

FIG. 2 illustrates TaqMan® assay results for assays designed from SEQ IDNO:1-6 to detect SEQ ID NO:111, in accordance with various embodiments.

FIG. 3 illustrates a seven by nucleotide insertion unique to SEQ IDNO:111.

FIG. 4 illustrates primer and probe sets useful in detecting SEQ IDNO:90-110.

FIG. 5 illustrates the results of assays used for the specific detectionof E. coli O157:H7 and not O55:H7.

DETAILED DESCRIPTION

For the purposes of interpreting of this specification, the followingdefinitions will apply and whenever appropriate, terms used in thesingular will also include the plural and vice versa. In the event thatany definition set forth below conflicts with the usage of that word inany other document, including any document incorporated herein byreference, the definition set forth below shall always control forpurposes of interpreting this specification and its associated claimsunless a contrary meaning is clearly intended (for example in thedocument where the term is originally used). It is noted that, as usedin this specification and the appended claims, the singular forms “a,”“an,” and “the,” include plural referents unless expressly andunequivocally limited to one referent. The use of “or” means “and/or”unless stated otherwise. The use of “comprise,” “comprises,”“comprising, “include,” “includes,” and “including” are interchangeableand not intended to be limiting. Furthermore, where the description ofone or more embodiments uses the term “comprising,” those skilled in theart would understand that, in some specific instances, the embodiment orembodiments can be alternatively described using the language“consisting essentially of” and/or “consisting of”

DEFINITIONS

As used herein, the phrase “nucleic acid,” “oligonucleotide”, andpolynucleotide(s)” are interchangeable and not intended to be limiting.

Reference will now be made to various embodiments, examples of which areillustrated in the accompanying drawings.

As used herein, the phrase “stringent hybridization conditions” refersto hybridization conditions which can take place under a number of pH,salt and temperature conditions. The pH can vary from 6 to 9, preferably6.8 to 8.5. The salt concentration can vary from 0.15 M sodium to 0.9 Msodium, and other cations can be used as long as the ionic strength isequivalent to that specified for sodium. The temperature of thehybridization reaction can vary from 30° C. to 80° C., preferably from45° C. to 70° C. Additionally, other compounds can be added to ahybridization reaction to promote specific hybridization at lowertemperatures, such as at or approaching room temperature. Among thecompounds contemplated for lowering the temperature requirements isformamide. Thus, a polynucleotide is typically “substantiallycomplementary” to a second polynucleotide if hybridization occursbetween the polynucleotide and the second polynucleotide. As usedherein, “specific hybridization” refers to hybridization between twopolynucleotides under stringent hybridization conditions.

As used herein, the term “polynucleotide” refers to a polymeric form ofnucleotides of any length, either ribonucleotides, deoxynucleotides, orpeptide nucleic acids (PNA), and includes both double- andsingle-stranded RNA, DNA, and PNA. A polynucleotide may includenucleotide sequences having different functions, including, forinstance, coding regions, and non-coding regions such as regulatoryregions. A polynucleotide can be obtained directly from a naturalsource, or can be prepared with the aid of recombinant, enzymatic, orchemical techniques. A polynucleotide can be linear or circular intopology. A polynucleotide can be, for example, a portion of a vector,such as an expression or cloning vector, or a fragment. An“oligonucleotide” refers to a polynucleotide of the present invention,typically a primer and/or a probe.

As used herein a “target-specific polynucleotide” refers to apolynucleotide having a target-binding segment that is perfectly orsubstantially complementary to a target sequence, such that thepolynucleotide binds specifically to an intended target withoutsignificant binding to non-target sequences under sufficiently stringenthybridization conditions. The target-specific polynucleotide can bee.g., a primer or probe and the subject of hybridization with itscomplementary target sequence.

The term “target sequence”, “target nucleic acid”, “target” or “targetpolynucleotide sequence” refers to a nucleic acid of interest. Thetarget sequence can be a polynucleotide sequence that is the subject ofhybridization with a complementary polynucleotide, e.g. a primer orprobe. The target sequence can be composed of DNA, RNA, an analogthereof, and including combinations thereof. The target sequence may beknown or not known, in terms of its actual sequence and itsamplification can be desired. The target sequence may or may not be ofbiological significance. Typically, though not always, it is thesignificance of the target sequence which is being studied in aparticular experiment. As non-limiting examples, target sequences mayinclude regions of genomic DNA, regions of genomic DNA which arebelieved to contain one or more polymorphic sites, DNA encoding orbelieved to encode genes or portions of genes of known or unknownfunction, DNA encoding or believed to encode proteins or portions ofproteins of known or unknown function, DNA encoding or believed toencode regulatory regions such as promoter sequences, splicing signals,polyadenylation signals, etc.

As used herein an “amplified target polynucleotide sequence product”refers to the resulting amplicon from an amplification reaction such asa polymerase chain reaction. The resulting amplicon product arises fromhybridization of complementary primers to a target polynucleotidesequence under suitable hybridization conditions and the repeating in acyclic manner the polymerase chain reaction as catalyzed by DNApolymerase for DNA amplification or RNA polymerase for RNAamplification.

As used herein, the “polymerase chain reaction” or PCR is a anamplification of nucleic acid consisting of an initial denaturation stepwhich separates the strands of a double stranded nucleic acid sample,followed by repetition of (i) an annealing step, which allowsamplification primers to anneal specifically to positions flanking atarget sequence; (ii) an extension step which extends the primers in a5′ to 3′ direction thereby forming an amplicon polynucleotidecomplementary to the target sequence, and (iii) a denaturation stepwhich causes the separation of the amplicon from the target sequence(Mullis et al., eds, The Polymerase Chain Reaction, BirkHauser, Boston,Mass. (1994). Each of the above steps may be conducted at a differenttemperature, preferably using an automated thermocycler (AppliedBiosystems LLC, a division of Life Technologies Corporation, FosterCity, Calif.). If desired, RNA samples can be converted to DNA/RNAheteroduplexes or to duplex cDNA by methods known to one of skill in theart.

As used herein, “amplifying” and “amplification” refers to a broad rangeof techniques for increasing polynucleotide sequences, either linearlyor exponentially. Exemplary amplification techniques include, but arenot limited to, PCR or any other method employing a primer extensionstep. Other nonlimiting examples of amplification include, but are notlimited to, ligase detection reaction (LDR) and ligase chain reaction(LCR). Amplification methods may comprise thermal-cycling or may beperformed isothermally. In various embodiments, the term “amplificationproduct” includes products from any number of cycles of amplificationreactions.

In certain embodiments, amplification methods comprise at least onecycle of amplification, for example, but not limited to, the sequentialprocedures of: hybridizing primers to primer-specific portions of targetsequence or amplification products from any number of cycles of anamplification reaction; synthesizing a strand of nucleotides in atemplate-dependent manner using a polymerase; and denaturing thenewly-formed nucleic acid duplex to separate the strands. The cycle mayor may not be repeated.

Descriptions of certain amplification techniques can be found, amongother places, in H. Ehrlich et al., Science, 252:1643-50 (1991), M.Innis et al., PCR Protocols: A Guide to Methods and Applications,Academic Press, New York, N.Y. (1990), R. Favis et al., NatureBiotechnology 18:561-64 (2000), and H. F. Rabenau et al., Infection28:97-102 (2000); Sambrook and Russell, Molecular Cloning, ThirdEdition, Cold Spring Harbor Press (2000) (hereinafter “Sambrook andRussell”), Ausubel et al., Current Protocols in Molecular Biology (1993)including supplements through September 2005, John Wiley & Sons(hereinafter “Ausubel et al.”).

The term “label” refers to any moiety which can be attached to amolecule and: (i) provides a detectable signal; (ii) interacts with asecond label to modify the detectable signal provided by the secondlabel, e.g. FRET; (iii) stabilizes hybridization, i.e. duplex formation;or (iv) provides a capture moiety, i.e. affinity, antibody/antigen,ionic complexation. Labelling can be accomplished using any one of alarge number of known techniques employing known labels, linkages,linking groups, reagents, reaction conditions, and analysis andpurification methods. Labels include light-emitting compounds whichgenerate a detectable signal by fluorescence, chemiluminescence, orbioluminescence (Kricka, L. in Nonisotopic DNA Probe Techniques (1992),Academic Press, San Diego, pp. 3-28). Another class of labels arehybridization-stabilizing moieties which serve to enhance, stabilize, orinfluence hybridization of duplexes, e.g. intercalators, minor-groovebinders, and cross-linking functional groups (Blackburn, G. and Gait, M.Eds. “DNA and RNA structure” in Nucleic Acids in Chemistry and Biology,2.sup.nd Edition, (1996) Oxford University Press, pp. 15-81). Yetanother class of labels effect the separation or immobilization of amolecule by specific or non-specific capture, for example biotin,digoxigenin, and other haptens (Andrus, A. “Chemical methods for 5′non-isotopic labelling of PCR probes and primers” (1995) in PCR 2: APractical Approach, Oxford University Press, Oxford, pp. 39-54).

The terms “annealing” and “hybridization” are used interchangeably andmean the base-pairing interaction of one nucleic acid with anothernucleic acid that results in formation of a duplex or otherhigher-ordered structure. The primary interaction is base specific, i.e.A/T and G/C, by Watson/Crick and Hoogsteen-type hydrogen bonding.

The term “end-point analysis” refers to a method where data collectionoccurs only when a reaction is substantially complete.

The term “real-time analysis” refers to periodic monitoring during PCR.Certain systems such as the ABI 7700 Sequence Detection System (AppliedBiosystems, Foster City, Calif.) conduct monitoring during each thermalcycle at a pre-determined or user-defined point. Real-time analysis ofPCR with FRET probes measures fluorescent dye signal changes fromcycle-to-cycle, preferably minus any internal control signals.

The term “quenching” refers to a decrease in fluorescence of a firstmoiety (reporter dye) caused by a second moiety (quencher) regardless ofthe mechanism.

A “primer,” as used herein, is an oligonucleotide that is complementaryto a portion of target polynucleotide and, after hybridization to thetarget polynucleotide, may serve as a starting-point for anamplification reaction and the synthesis of an amplification product.Primers include, but are not limited to, spanning primers. A “primerpair” refers to two primers that can be used together for anamplification reaction. A “PCR primer” refers to a primer in a set of atleast two primers that are capable of exponentially amplifying a targetnucleic acid sequence in the polymerase chain reaction.

The term “probe” comprises a polynucleotide that comprises a specificportion designed to hybridize in a sequence-specific manner with acomplementary region of a specific nucleic acid sequence, e.g., a targetnucleic acid sequence. In certain embodiments, the specific portion ofthe probe may be specific for a particular sequence, or alternatively,may be degenerate, e.g., specific for a set of sequences. In certainembodiments, the probe is labeled. The probe can be an oligonucleotidethat is complementary to at least a portion of an amplification productformed using two primers.

The terms “complement” and “complementary” as used herein, refer to theability of two single stranded polynucleotides (for instance, a primerand a target polynucleotide) to base pair with each other, where anadenine on one strand of a polynucleotide will base pair to a thymine oruracil on a strand of a second polynucleotide and a cytosine on onestrand of a polynucleotide will base pair to a guanine on a strand of asecond polynucleotide. Two polynucleotides are complementary to eachother when a nucleotide sequence in one polynucleotide can base pairwith a nucleotide sequence in a second polynucleotide. For instance,5′-ATGC and 5′-GCAT are complementary.

A “label” refers to a moiety attached (covalently or non-covalently), orcapable of being attached, to an oligonucleotide, which provides or iscapable of providing information about the oligonucleotide (e.g.,descriptive or identifying information about the oligonucleotide) oranother polynucleotide with which the labeled oligonucleotide interacts(e.g., hybridizes). Labels can be used to provide a detectable (andoptionally quantifiable) signal. Labels can also be used to attach anoligonucleotide to a surface.

A “fluorophore” is a moiety that can emit light of a particularwavelength following absorbance of light of shorter wavelength. Thewavelength of the light emitted by a particular fluorophore ischaracteristic of that fluorophore. Thus, a particular fluorophore canbe detected by detecting light of an appropriate wavelength followingexcitation of the fluorophore with light of shorter wavelength.

The term “quencher” as used herein refers to a moiety that absorbsenergy emitted from a fluorophore, or otherwise interferes with theability of the fluorescent dye to emit light. A quencher can re-emit theenergy absorbed from a fluorophore in a signal characteristic for thatquencher, and thus a quencher can also act as a fluorophore (afluorescent quencher). This phenomenon is generally known as fluorescentresonance energy transfer (FRET). Alternatively, a quencher candissipate the energy absorbed from a fluorophore as heat (anon-fluorescent quencher).

As used herein, a “sample” refers to any substance comprising nucleicacid material. A sample to be tested and can include food intended forhuman or animal consumption such as meat, nuts, legumes, fruit, andvegetables, a beverage sample, a fermentation broth, a forensic sample,an environmental sample (e.g., soil, dirt, garbage, sewage, air, orwater), including food processing and manufacturing surfaces, or abiological sample. A “biological sample” refers to a sample obtainedfrom eukaryotic or prokaryotic sources. Examples of eukaryotic sourcesinclude mammals, such as a human or a cow or a member of the familyMuridae (a murine animal such as rat or mouse). Alternatively, thesample may include blood, urine, feces, or other materials from a humanor a livestock animal. The sample may be tested directly, or may betreated in some manner prior to testing. For example, the sample may besubjected to PCR amplification using appropriate oligonucleotideprimers. Examples of prokaryotic sources include enterococci. Thebiological sample can be, for instance, in the form of a single cell, inthe form of a tissue, or in the form of a fluid.

As used herein, “detecting” or “detection” refers to the disclosure orrevelation of the presence or absence in a sample of a targetpolynucleotide sequence or amplified target polynucleotide sequenceproduct. The detecting can be by end point, real-time, enzymatic, and byresolving the amplification product on a gel and determining whether theexpected amplification product is present, or other methods known to oneof skill in the art.

The presence or absence of an amplified product can be determined or itsamount measured. Detecting an amplified product can be conducted bystandard methods well known in the art and used routinely. The detectingmay occur, for instance, after multiple amplification cycles have beenrun (typically referred to an end-point analysis), or during eachamplification cycle (typically referred to as real-time). Detecting anamplification product after multiple amplification cycles have been runis easily accomplished by, for instance, resolving the amplificationproduct on a gel and determining whether the expected amplificationproduct is present. In order to facilitate real-time detection orquantification of the amplification products, one or more of the primersand/or probes used in the amplification reaction can be labeled, andvarious formats are available for generating a detectable signal thatindicates an amplification product is present. For example, a convenientlabel is typically a label that is fluorescent, which may be used invarious formats including, but are not limited to, the use of donorfluorophore labels, acceptor fluorophore labels, fluorophores,quenchers, and combinations thereof. Assays using these various formatsmay include the use of one or more primers that are labeled (forinstance, scorpions primers, amplifluor primers), one or more probesthat are labeled (for instance, adjacent probes, TaqMan® probes,light-up probes, molecular beacons), or a combination thereof. Theskilled person will understand that in addition to these known formats,new types of formats are routinely disclosed. The present invention isnot limited by the type of method or the types of probes and/or primersused to detect an amplified product. Using appropriate labels (forexample, different fluorophores) it is possible to combine (multiplex)the results of several different primer pairs (and, optionally, probesif they are present) in a single reaction. As an alternative todetection using a labeled primer and/or probe, an amplification productcan be detected using a polynucleotide binding dye such as a fluorescentDNA binding dye. Examples include, for instance, SYBR® Green dye orSYBR® Gold dye (Molecular Probes). Upon interaction with thedouble-stranded amplification product, such polynucleotide binding dyesemit a fluorescence signal after excitation with light at a suitablewavelength. A polynucleotide binding dye such as a polynucleotideintercalating dye also can be used.

As used herein, an “E. coli O157:H7-specific nucleotide probe” refers toa sequence that is able to specifically hybridize to an E. coli O157:H7target sequence present in a sample containing E. coli O157:H7 undersuitable hybridization conditions and which does not hybridize with DNAfrom other E. coli strains or from other bacterial species. It is wellwithin the ability of one skilled in the art to determine suitablehybridization conditions based on probe length, G+C content, and thedegree of stringency required for a particular application.

It is expected that minor sequence variations in E. coliO157:H7-specific nucleotide sequences associated with nucleotideadditions, deletions and mutations, whether naturally occurring orintroduced in vitro, would not interfere with the usefulness of SEQ IDNO:1-111 in the detection of enterohemorrhagic E. coli (EHEC), inmethods for preventing EHEC infection, and in methods for treating EHECinfection, as would be understood by one of skill in the art. Therefore,the scope of the present invention as claimed is intended to encompassminor variations in the sequences of SEQ ID NO:1-111 and sequenceshaving at least 90% homology to the SEQ ID NO:1-111 sequences.

The probe may be RNA or DNA. Depending on the detection means employed,the probe may be unlabeled, radiolabeled, chemiluminescent labeled,enzyme labeled, or labeled with a dye. The probe may be hybridized witha sample in solution or immobilized on a solid support such asnitrocellulose, a microarray or a nylon membrane, or the probe may beimmobilized on a solid support, such as a silicon chip or a microarray.

Conditions that “allow” an event to occur or conditions that are“suitable” for an event to occur, such as hybridization, strandextension, and the like, or “suitable” conditions are conditions that donot prevent such events from occurring. Thus, these conditions permit,enhance, facilitate, and/or are conducive to the event. Such conditions,known in the art and described herein, may depend upon, for example, thenature of the nucleotide sequence, temperature, and buffer conditions.These conditions may also depend on what event is desired, such ashybridization, cleavage, or strand extension. An “isolated”polynucleotide refers to a polynucleotide that has been removed from itsnatural environment. A “purified” polynucleotide is one that is at leastabout 60% free, preferably at least about 75% free, and most preferablyat least about 90% free from other components with which they arenaturally associated.

The words “preferred” and “preferably” refer to embodiments of theinvention that may afford certain benefits, under certain circumstances.However, other embodiments may also be preferred, under the same orother circumstances. Furthermore, the recitation of one or morepreferred embodiments does not imply that other embodiments are notuseful, and is not intended to exclude other embodiments from the scopeof the invention.

The terms “comprises” and variations thereof do not have a limitingmeaning where these terms appear in the description and claims. Unlessotherwise specified, “a,” “an,” “the,” and “at least one” are usedinterchangeably and mean one or more than one.

Also herein, the recitations of numerical ranges by endpoints includeall numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2,2.75, 3, 3.80, 4, 5, etc.). The term “and/or” means one or all of thelisted elements or a combination of any two or more of the listedelements.

There are many known methods of amplifying nucleic acid sequencesincluding e.g., PCR. See, e.g., PCR Technology: Principles andApplications for DNA Amplification (ed. H. A. Erlich, Freeman Press, NY,N.Y., 1992); PCR Protocols: A Guide to Methods and Applications (eds.Innis, et al., Academic Press, San Diego, Calif., 1990); Mattila et al.,Nucleic Acids Res. 19, 4967 (1991); Eckert et al., PCR Methods andApplications 1, 17 (1991); PCR (eds. McPherson et al., IRL Press,Oxford); and U.S. Pat. Nos. 4,683,202, 4,683,195, 4,800,159 4,965,188and 5,333,675 each of which is incorporated herein by reference in theirentireties for all purposes.

Nucleic acid amplification techniques are traditionally classifiedaccording to the temperature requirements of the amplification process.Isothermal amplifications are conducted at a constant temperature, incontrast to amplifications that require cycling between high and lowtemperatures. Examples of isothermal amplification techniques are:Strand Displacement Amplification (SDA; Walker et al., 1992, Proc. Natl.Acad. Sci. USA 89:392 396; Walker et al., 1992, Nuc. Acids. Res. 20:16911696; and EP 0 497 272, all of which are incorporated herein byreference), self-sustained sequence replication (3SR; Guatelli et al.,1990, Proc. Natl. Acad. Sci. USA 87:1874 1878), the Q.beta. replicasesystem (Lizardi et al., 1988, BioTechnology 6:1197 1202), and thetechniques disclosed in WO 90/10064 and WO 91/03573.

Examples of techniques that require temperature cycling are: polymerasechain reaction (PCR; Saiki et al., 1985, Science 230:1350 1354), ligasechain reaction (LCR; Wu et al., 1989, Genomics 4:560 569; Barringer etal., 1990, Gene 89:117 122; Barany, 1991, Proc. Natl. Acad. Sci. USA88:189 193), transcription-based amplification (Kwoh et al., 1989, Proc.Natl. Acad. Sci. USA 86:1173 1177) and restriction amplification (U.S.Pat. No. 5,102,784).

Other exemplary techniques include Nucleic Acid Sequence-BasedAmplification (“NASBA”; see U.S. Pat. No. 5,130,238), Q.beta. replicasesystem (see Lizardi et al., BioTechnology 6:1197 (1988)), and RollingCircle Amplification (see Lizardi et al., Nat Genet. 19:225 232 (1998)).The amplification primers of the present invention may be used to carryout, for example, but not limited to, PCR, SDA or tSDA. Any of theamplification techniques and methods disclosed herein can be used topractice the claimed invention as would be understood by one of ordinaryskill in the art.

PCR is an extremely powerful technique for amplifying specificpolynucleotide sequences, including genomic DNA, single-stranded cDNA,and mRNA among others. Various methods of conducting PCR amplificationand primer design and construction for PCR amplification will be knownto those of skill in the art. Generally, in PCR a double-stranded DNA tobe amplified is denatured by heating the sample. New DNA synthesis isthen primed by hybridizing primers to the target sequence in thepresence of DNA polymerase and excess dNTPs. In subsequent cycles, theprimers hybridize to the newly synthesized DNA to produce discreetproducts with the primer sequences at either end. The productsaccumulate exponentially with each successive round of amplification.

The DNA polymerase used in PCR is often a thermostable polymerase. Thisallows the enzyme to continue functioning after repeated cycles ofheating necessary to denature the double-stranded DNA. Polymerases thatare useful for PCR include, for example, Taq DNA polymerase, Tth DNApolymerase, Tfl DNA polymerase, Tma DNA polymerase, Tli DNA polymerase,and Pfu DNA polymerase. There are many commercially available modifiedforms of these enzymes including: AmpliTaq® and AmpliTaq Gold® bothavailable from Applied Biosystems. Many are available with or without a3- to 5′ proofreading exonuclease activity. See, for example, Vent® andVent®. (exo-) available from New England Biolabs.

Other suitable amplification methods include the ligase chain reaction(LCR) (e.g., Wu and Wallace, Genomics 4, 560 (1989) and Landegren etal., Science 241, 1077 (1988)), transcription amplification (Kwoh etal., Proc. Natl. Acad. Sci. USA 86, 1173 (1989)), and self-sustainedsequence replication (Guatelli et al., Proc. Nat. Acad. Sci. USA, 87,1874 (1990)) and nucleic acid based sequence amplification (NABSA).(See, U.S. Pat. Nos. 5,409,818, 5,554,517, and 6,063,603). The lattertwo amplification methods include isothermal reactions based onisothermal transcription, which produce both single-stranded RNA (ssRNA)and double-stranded DNA (dsDNA) as the amplification products in a ratioof about 30 or 100 to 1, respectively.

Amplicon Selection:

Detection of E. coli O157:H7 by the use of the polymerase chain reactionprovides a rapid method for detection. Moreover, the primer(s) (andprobe used in a real-time PCR reaction) is desired to be specific andsensitive for only the organism of interest, i.e., E. coli O157:H7.However, the genome of E. coli O157:H7 is very similar to other E. coligenomes, both those which are disease causing (i.e., infectious) andpathogenic (ability to cause damage) e.g., serotype O55:H7 and thosewhich are not pathogenic, e.g., serotype K12. Therefore, in oneembodiment, the identification and selection of genomic sequence from E.coli O157:H7 (e.g., strain EDL993, GenBank Accession No. AE005174,Perna, N. T., et al., (2001) Nature 409(25):529-533) for the design ofreal-time PCR assays is based on the differential identification of E.coli O157:H7 genomic sequence (I, inclusion set) not found in closelyrelated strains of E. coli (E, exclusion set). By identifying sequencesnot found in both infections and non-infectious strains of E. coli,target sequences specific to E. coli O157:H7 can be identified forprimer design that do not, or only with rare exception, detect closelyrelated E. coli strains and therefore identify E. coli O157:H7 withspecificity and sensitivity.

Identification of E. coli O157:H7 Unique Sequence Regions:

The E. coli O157:H7 strain EDL933 genome has about 5.6 million basepairs (Mb) of DNA in the backbone sequence. When compared to a common,non-pathogenic laboratory strain, E. coli K12 strain MG1655, havingabout 4.6 Mb of DNA. Each genome has regions of DNA unique to one strainor the other, numbering in the hundreds of regions.

Prior to the claimed invention ‘O-islands’ where described as nucleicacid sequence regions unique to and found only in the O157:H7 serotype.O-islands regions total 1.34 megabases and 1,387 genes whereas‘K-islands’ are described as being unique to serotype K12, totaling 0.53megabases and 528 genes. (Perna, N. T., et al., supra). The design ofassays specific for the O157:H7 serotype as described herein refutes theuniqueness of O-islands to only the O157:H7 serotype. Comparison of thegenome of O157:H7 with that of O55:H7 unexpectedly revealed that theO55:H7 serotype also contained O-islands virtually identical to those ofO157:H7. Therefore, prior to the claimed invention, a definitiveidentification of O157:H7 was difficult due to genome sequencesimilarity, even in supposedly serotype specific regions. The presentinvention as claimed has identified serotype unique DNA sequences for E.coli O157:H7 which were utilized for assay design and the subsequentdetection of E. coli O157:H7 and not O55:H7 by PCR, hybridization andother molecular biology techniques as known to one skilled in the art.

The sequence of the E. coli O157:H7 genome is represented in publicdatabases such as GenBank (NCBI, National Library of Medicine, TheNational Institutes of Health, Bethesda, Md.) EMBL, and DDBJ. One ofskill in the art quickly recognizes that the genomic sequence of O157:H7serotype is representative of a variety of non-O157:H7, yet pathogenicserotypes such as O111:H7, 026:H11 and O103:H2. A bioinformatic approachto identify E. coli O157:H7 unique sequence regions was undertaken andevaluated by Sanger sequencing and real-time PCR assays.

In one embodiment of the current teachings bioinformatic and direct DNAsequencing comparisons were conducted in an effort to identify E. coliO157:H7 serotype-specific sequences. 28 separate E. coli O157:H7serotype EDL933 (GenBank Acc No. AE005174) O-island sequence regionswere downloaded from the NCBI database GenBank, release 160). TheseO-islands were known to be present, absent in some or variants ofO-islands in E. coli O157:H7. Additional O-island and K-island regionswere sequenced by the Sanger/capillary electrophoresis method known toone of skill in the art from the Applied Biosystems microbial DNAcollection of E. coli serotypes O157:H7, K12 and others. Alignment ofthe sequenced regions using algorithms known to one of ordinary skill inthe art identified no O157:H7 “unique” regions. PCR primer pairs weredesigned for each of the 28 regions to specifically amplify the uniquesequences within the O-islands against both inclusion (organism to bedetected, i.e., E. coli O157:H7) and exclusion genomes (organisms not tobe detected, i.e., E. coli non-O157:H7 serotypes and Shigella spp.)within the Applied Biosystems microbial DNA collection. The resultingamplification products were then sequenced and aligned to the EDL933genome. Comparison of the alignments of the O157:H7 specific regions tonon-O157:H7 sequences identified an E. coli O157:H7 sequence region (SEQID NO:111) that was used for the identification of E. coli O157:H7antigen specific strains. PCR primer pairs (and probes for use inreal-time PCR assays) were designed to the O157:H7 specific region andscreened against ground beef. Any assay having a positive resultindicated the assay cross-reacted with ground beef and was removed fromfurther analyses. As shown in FIG. 1, the remaining PCR primer pairs andprobes (SEQ ID NOs:1-6) were used in real-time PCR assays and evaluatedagainst the Applied Biosystems microbial DNA collection (FIG. 2). Forexample, primer pairs SEQ ID NO:1-2, SEQ ID NO:1 and SEQ ID NO:4, SEQ IDNO:1 and SEQ ID NO:5, and SEQ ID NO:1 and SEQ ID NO:6 can be used forend-point analysis and when used in conjunction with SEQ ID NO:3 as theprobe comprised the real-time PCR assays as listed in FIG. 1.

The primers and probes from FIG. 1 were used in real-time PCR assays onDNA extracts from various strains from the Applied Biosystems microbialDNA collection and have experimentally demonstrated the specificity ofeach assay for identification of E. coli O157:H7 and a weak signal forE. coli O55:H7 (FIG. 2), an unexpected result. A C_(t) value >35 wasconsider a “weak positive” result. When a C_(t) value is present asignal was detected, a positive result and ‘no signal’ indicates nodetectable signal, and so a negative result. In all selected serotypesshown, the internal positive control had a detectable signal/positiveresult (data not shown).

As shown in FIG. 3, this region of the O157:H7 genome has a sevenbasepair insertion (a direct repeat) that is not present in O55:H7. Thisseven basepair insertion in O157:H7 relative to O55:H7 is the onlysignificant sequence difference found between the two serotypes in SEQID NO:111. The forward primer of assay 18158 overlaps this insertion,which provides some specificity for O157:H7. Analysis of GenBanksequences for the SEQ ID NO:111 region indicted that the full-lengthsequence of SEQ ID 111 is found only in E. coli O157:H7, Shigellasonnei, and Shigella boydii. In all cases except O157:H7, the sevennucleotide O157:H7-specific insertion is absent. Other fragments of SEQID 111 that are more widely conserved among E. coli are nucleotidesequence positions 355 to 456, and 156 to 238. The 355-456 fragmentoverlaps the reverse primer of assay 18158, and partially overlaps theprobe, but not the forward primer, which can account for the 18158 assaynot detecting more serotypes.

These assays have been shown to be very specific for the rapid detectionof E. coli O157:H7 and weakly positive for E. coli O55:H7 from isolatedDNA and from ground beef samples. Experimental results confirmed thatthese assays have 100% sequence identity to the O157:H7 genome.Bioinformatic evidence indicated that the assays were specific forO157:H7 EDL933 and O157:H7 Sakai genomes, GenBank Accession Nos.AE005174 and NC_(—)002695.1, respectively.

In another embodiment of the current teachings bioinformatic and directDNA sequencing comparisons were conducted in an effort to identifysequences common to E. coli O157:H7 strains but absent or highlydivergent in E. coli strains within the exclusion set. 14 E. coliO157:H7 genome sequences and 30 non-O157:H7 E. coli and Shigella spp.were downloaded from public databases (NCBI database GenBank, release161.0, or the Sanger Center's public FTP site (Wellcome Trust SangerInstitute). The bioinformatic approach entailed aligning each of the 45sequences against an E. coli O157:H7 reference genome (GenBank AccessionNo. NC_(—)002695.1). The alignments and parsed results from the initialgenome comparisons resulted in identifying 157 O157:H7 unique regions.These regions were then analyzed by BLASTN against the GenBanknon-redundant database (nr). Sequences with at least 80% similarity over50 or more contiguous nucleotides were removed from further analysis. Ofthose removed about two thirds of hits with more than 85% sequenceidentity were from strains of E. coli, Shigella or E. fergusonii forwhich whole genome sequence was not available. The remain ⅓ were toEnterobacter, Salmonella or Erwina. The resulting set of 118 O157:H7unique regions were considered target sequences totaling 117 kb. Assayswere designed from unique E. coli O157:H7 sequence regions which wereidentified by alignment of genomes.

The nearest neighbor of E. coli O157:H7 is E. coli O55:H7. Oursequencing in E. coli O55:H7 strains for known E. coli O157:H7 O-islandsfurther supported the close relationship between the two serotypes asmost O-islands were conserved between the two serotypes. Further genomicsequencing of the O55:H7 genome (PE704 isolate) in parallel with thesequencing of an O157:H7 strain (PE30 isolate) also supported the veryclose relationship of the two serotypes as single nucleotidepolymorphisms (SNPs) in O55:H7 verse O157:H7 was only 0.28% (data notshown).

The 118 target sequences for O157:H7 were screened against the O55:H7consensus genome assembly using BLASTN. Only 9.9 kb or 8.5% of thecandidate O157:H7-specific sequence identified by genome comparison wasfound to be absent from the O55:H7 genome. 20 target sequences werefound to be absent from the O55:H7 genome and all 20 had at least 98.85%sequence identity between any pair of the 15 publicly available O157:H7genomes indicating each target sequence was highly conserved and assaysdesigned from these regions would be highly indicative of E. coliO157:H7 strains and not O55:H7.

The 20 target sequences corresponded to seven regions identified in U.S.Pat. No. 6,365,723, incorporated herein by reference in its entirety. Asindicated in Table 1, these regions are associated with CP933-M,N,X andP, cryptic prophages, each having a collection of open reading frames,and fimbrial proteins. For example, primer pairs SEQ ID NO:7-8, SEQ IDNO:10-11, SEQ ID NO:13-14, SEQ ID NO:16-17, SEQ ID NO:19-20, SEQ IDNO:22-23, SEQ ID NO:25-26, SEQ ID NO:28-29, SEQ ID NO:31-32, SEQ IDNO:34-35, SEQ ID NO:37-38, SEQ ID NO:40-41, SEQ ID NO:43-44, SEQ IDNO:46-47, SEQ ID NO:49-50, SEQ ID NO:52-53, SEQ ID NO:55-56, SEQ IDNO:59 and SEQ ID NO:56, SEQ ID NO:61-62, SEQ ID NO:64-65, SEQ IDNO:67-68, SEQ ID NO:70-71, SEQ ID NO:73-74, SEQ ID NO:76-77, SEQ IDNO:79-80, SEQ ID NO:82-83, SEQ ID NO:85-86, and SEQ ID NO:88-89 can beused for end-point analysis and when used in conjunction with a probe ofSEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21, SEQID NO:24, SEQ ID NO:27 SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ IDNO:39, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ IDNO:54, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:63, SEQ IDNO:66, SEQ ID NO:69, SEQ ID NO:72, SEQ ID NO:75, SEQ ID NO:78, SEQ IDNO:81, SEQ ID NO:84, SEQ ID NO:87, or SEQ ID NO:90 as listed in FIG. 4,comprise real-time PCR assays. These 20 target sequences (SEQ IDNO:91-110) were used in the design of O157:H7-specific real-time PCRassays using algorithms known by one of skill in the art (FIG. 4). The29 assays map to the seven regions as indicated in Table 1.

TABLE 1 E. coli O157:H7-specific genomic regions^(a) Region EDL933EDL933 Length number start end (nt) ORFs Features 1 1255352 1256121 770Z1328, Z1329 CP933-M 2 1256573 1257839 1267 Z1332-Z1334 CP933-M 31262147 1263665 1519 Z1341 CP933-M  4* 1635627 1635757 131 Z1781 CP933-N5 1744789 1747582 2794 Z1921-Z1924 CP933-X  6* 2319451 2319578 128 Z6065CP933-P 7 2933425 2934070 646 Z3276 Fimbrial protein ^(a)as mapped toGenBank Acc. No. AE005174 *Regions 4 and 6 are duplicate copies of thesame sequence.

Real-time PCR assays were designed from seven unique and specific E.coli O157:H7 sequence regions. The assays were then tested against theApplied Biosystems microbial DNA collection of E. coli strains andserotypes to confirm the specificity, sensitivity and unambiguousdetection of only E. coli O157:H7 nucleic acid. However, unexpectedly,the selected assays also amplified nucleic acid from one strain of E.coli O103:H2 serotype and only one of four strains of the E. coli026:H11 serotype. Subsequent testing revealed that the positive resultsfor only one E. coli 026:H11 sample indicated mistyping of the sample.

In one embodiment, combining the results of an assay that detects atarget polynucleotide sequence of SEQ ID NO:111 with the results of anassay that detects a target polynucleotide sequence selected from SEQ IDNO:91-110 provides specific and definitive detection for E. coli O157:H7and not O55:H7. As shown in Table 2, the combination of assay Nos. 18158and 19055 whether in single or duplex assay format provides anunambiguous positive test for E. coli O157:H7, i.e., a positive test forO157:H7 in each assay confirms a positive test for E. coli O157:H7.

TABLE 2 Assay No. Assay No. Interpreted Serotype 18158 19055 resultO157:H7 (all) + + Positive O55:H7 (all) weak+ − − O103:H2 (1/1) − + −O26:H11 (1/4) − + −

Clearly, neither assay alone is definitive for a single serotype of E.coli due to genomic similarity between the genomic regions of other E.coli serotypes. Yet, when two assays, as example, but not limited to theassays of Table 2, are used either in parallel or as a multiplex assay,e.g., in a real-time TaqMan® assay, for example, where each probe ineach of the two assays has a different label for distinguishing resultson a real-time PCR instrument, e.g., a 7500 Fast Real-Time PCR System(Applied Biosystems), a positive result from each assay is indicative ofonly E. coli O157:H7. Thus, without knowledge of genomic regions sharedby serotype O157:H7 and O55:H7, design of an unambiguous, specific andsensitive test for E. coli O157:H7 would not be possible.

The claimed method to identify E. coli O157:H7 results from atwo-pronged approach to identify target sequences for O157:H7 that donot also detect closely related either pathogenic or non-pathogenic E.coli. The identified E. coli O157:H7 target sequences were used todesign primers and probes for real-time PCR assays. Programs known toone of skill in the art for assay design include Primer3 (Steve Rozenand Helen J. Skaletsky (2000) “Primer3” on the World Wide Web forgeneral users and for biologist programmers as published in: Krawetz S,Misener S (eds) Bioinformatics Methods and Protocols: Methods inMolecular Biology. Humana Press, Totowa, N.J., pp 365-386), PrimerExpress® software (Applied Biosystems), and OLIGO 7 (Wojciech Rychlik(2007). “OLIGO 7 Primer Analysis Software”. Methods Mol. Biol. 402:35-60). The subsequently designed PCR primers and probes for use inassays by real-time PCR detected unambiguously, specifically and withgreat sensitivity E. coli O157:H7 and not O55:H7.

An assay from FIG. 2 along with an assay from FIG. 3 either in the samereaction vessel as a duplex (multiplex) assay or in separate reactionvessels have experimentally demonstrated the specificity of thedual-assay approach for identification of E. coli O157:H7 and theability to distinguish O157:H7 from E. coli O55:H7 (Example 2). Theresults were also confirmed by sequencing various isolates of bothserotypes O157:H7 and O55:H7. As indicated in FIG. 5, the results ofreal-time PCR analyses on DNA extracts from various matrix samples andvarious strains from the Applied Biosystems microbial DNA collection.When a C_(t) value is present a signal was detected, a positive resultand ‘no result’ indicates no signal detected, a negative result. A C_(t)value >35 was determined to be a “weak positive”. Both assays having adetected signal for O157:H7 were a positive identification of E. coliO157:H7. When at least one or both assays have no signal for O157:H7,the result is considered negative for E. coli O157:H7. In all selectedserotypes shown, the internal positive control had a detectablesignal/positive result.

The rare signal detected from a serotype other than E. coli O157:H7 wasunexpected for the dual/multiplex assay. The likelihood of thesimultaneous presence of a rare serotype being mistaken for E. coliO157:H7 is extremely remote. Requiring a positive result for eachreal-time assay as seen by a different detectable label on each probefor discrimination between each assay's amplification reaction providesa result that would be understood by the skilled artisan to beunambiguous, specific and sensitive for the detection of E. coliO157:H7.

The dual or multiplex (more than 2 assay sets) assay approach can beused to detect and distinguish other regional pathogenic E. coli. EHECis identified as being caused by two classes of E. coli. Class 1 ispredominant in the United States, caused by pathogenic strains of E.coli O157:H7 and Class 2 is more geographically dispersed and is causedby E. coli O26 and E. coli O111. As provided in Table 3, depending onthe local beef population's indigenous E. coli and possible importedbeef source, various pathogenic strains of E. coli are a public healththreat.

TABLE 3 Country/Continent E. coli Serotye (s) Class United StatesO157:H7 1 South America O26 and O111 2 Scotland O26 and O111 2 GermanyO157:H7 and O26 1 & 2 Australia O26 and O111 2

The above summary of the present invention is not intended to describeeach disclosed embodiment or every implementation of the presentinvention. The description that follows more particularly exemplifiesillustrative embodiments. In several places throughout the application,guidance is provided through lists of examples, which examples can beused in various combinations. In each instance, the recited list servesonly as a representative group and should not be interpreted as anexclusive list.

The materials for use in the present invention are ideally suited forthe preparation of a kit suitable for identifying the presence of E.coli O157:H7 and not E. coli O55:H7. Such a kit may comprise variousreagents utilized in the methods, preferably in concentrated form. Thereagents of this kit may comprise, but are not limited to, buffer,appropriate nucleotide triphosphates, DNA polymerases, intercalatingdye, primers, probes, salt, and instructions for the use of the kit.

Those having ordinary skill in the art will understand that manymodifications, alternatives, and equivalents are possible. All suchmodifications, alternatives, and equivalents are intended to beencompassed herein.

EXAMPLES

The following procedures are representative of procedures that can beemployed for the detection of E. coli O157:H7.

Example 1 PCR Reaction Conditions to Evaluate O-Island Assays

All amplifications are done with “TrueAllele” PCR Master MixPrimer pairs prepped at a [5 μM] eachTemplate is 1 μL, of a 1→100 dilution of the bacterial lysate

Reaction Mix Preparation:

Per reaction for 50 reactions True Allele MM 18 μl  900 μl Primer mix 5μM 2 μl 100 μl ddH₂O 9 μl 450 μl vortex briefly to mix components

Plate Preparation:

-   -   Pipette 1 μl of template from the template plate to reaction        plate conserving well position.    -   Add 29 μl of Reaction Mix    -   Seal with adhesive cover, staking cover very well    -   Vortex briefly to mix components    -   Spin down plates and thermalcycle

Thermal cycler profile: 95° C. 10′→95° C. 15″−60° C. 60″ (×30 cycles)→4°C.

Example 2 Sequencing Reaction Protocol

Sequencing Reaction Mix:

Per reaction for 100 reactions 5x sequencing buffer 3.2 320 BDT v.3.1 RRmix 1.6 160 Seq. primer 1 μM 1.0 100 ddH2O 7.2 720 total 13 1300

-   1. Add 13 μl of above reaction mix to 7 μl of Exo-SAPped PCR    amplicon, vortex, spin down and thermal cycle

Dye Exterminator Clean-Up of Sequencing Reactions Protocol

-   1. Prepare Dye Exterminator Mix:    -   82% SAM buffer    -   18% Dye Exterminators beads (agitate before pipetting)

For 96 sequences prepare 10 ml: 8.2 ml of SAM+1.8 ml of beads

-   2. For each 10 μl of sequencing reactions add 50 μl of Dye    Exterminator mix: to each 20 μl reaction add 100 μl-   3. Heat seal plates at 160° C. for 2 seconds-   4. Vortex at ˜1800 rpm for 30 minutes-   5. Spin down in centrifuge at ˜1000 rpm for 2 minutes-   6. Load plates on sequencer and make sure to use appropriate Dye    Exterminator module for injection

While the foregoing specification teaches the principles of the presentinvention, with examples provided for the purpose of illustration, itwill be appreciated by one skilled in the art from reading thisdisclosure that various changes in form and detail can be made withoutdeparting from the spirit and scope of the invention. These methods arenot limited to any particular type of nucleic acid sample: plant,bacterial, animal (including human) total genome DNA, RNA, cDNA and thelike may be analyzed using some or all of the methods disclosed in thisinvention. This invention provides a powerful tool for analysis ofcomplex nucleic acid samples. From experiment design to detection of E.coli O157:H7 assay results, the above invention provides for fast,efficient and inexpensive methods for detection of pathogenic E. coliO157:H7.

All publications and patent applications cited above are incorporated byreference in their entirety for all purposes to the same extent as ifeach individual publication or patent application were specifically andindividually indicated to be so incorporated by reference. Although thepresent invention has been described in some detail by way ofillustration and example for purposes of clarity and understanding, itwill be apparent that certain changes and modifications may be practicedwithin the scope of the appended claims.

1. A method of detecting the presence of E. coli O157:H7 in a sample,comprising: a.) detecting the presence of SEQ ID NO:111 or complementthereof; and b.) detecting the presence of a sequence selected from SEQID NO:90-110 and complements thereof; wherein detection of SEQ ID NO:111and detection of a sequence selected from SEQ ID NO:90-110 confirms thepresence of E. coli O157:H7 in a sample and not E. coli O55:H7.
 2. Themethod of claim 1, wherein the detection is by a nucleic acidamplification reaction.
 3. The method of claim 2, wherein theamplification reaction is an end-point determination.
 4. The method ofclaim 2, wherein the amplification reaction is quantitative.
 5. Themethod of claim 4, wherein the quantification is a real-time PCR.
 6. Themethod of claim 5, wherein the real-time PCR is a SYBR® Green Assay. 7.The method of claim 5, wherein the real-time PCR is a TaqMan® Assay. 8.An assay for the detection of E. coli O157:H7 in a sample comprising: a)hybridizing a first pair of PCR primers selected from the groupconsisting of: SEQ ID NO:1-2, SEQ ID NO:1 and SEQ ID NO:4, SEQ ID NO:1and SEQ ID NO:5, and SEQ ID NO:1 and SEQ ID NO:6 and complements thereofto at least a first target polynucleotide sequence; b) hybridizing asecond pair of PCR primers selected from SEQ ID NO:7-8, SEQ ID NO:10-11,SEQ ID NO:13-14, SEQ ID NO:16-17, SEQ ID NO:19-20, SEQ ID NO:22-23, SEQID NO:25-26, SEQ ID NO:28-29, SEQ ID NO:31-32, SEQ ID NO:34-35, SEQ IDNO:37-38, SEQ ID NO:40-41, SEQ ID NO:43-44, SEQ ID NO:46-47, SEQ IDNO:49-50, SEQ ID NO:52-53, SEQ ID NO:55-56, SEQ ID NO:59 and SEQ IDNO:56, SEQ ID NO:61-62, SEQ ID NO:64-65, SEQ ID NO:67-68, SEQ IDNO:70-71, SEQ ID NO:73-74, SEQ ID NO:76-77, SEQ ID NO:79-80, SEQ IDNO:82-83, SEQ ID NO:85-86, and SEQ ID NO:88-89 and complements thereofto at least a second target polynucleotide sequence; c) amplifying saidat least first and said at least second target polynucleotide sequences;and d) detecting said at least first and said at least second amplifiedtarget polynucleotide sequence products; wherein the detection of the atleast first amplified target polynucleotide sequence product and thedetection of the at least second amplified target polynucleotidesequence product is indicative of the presence of E. coli O157:H7 in thesample and not E. coli O55:H7.
 9. The assay of claim 8, furthercomprising a first probe of SEQ ID NO:3 and a second probe selected fromSEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21, SEQID NO:24, SEQ ID NO:27 SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ IDNO:39, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ IDNO:54, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:63, SEQ IDNO:66, SEQ ID NO:69, SEQ ID NO:72, SEQ ID NO:75, SEQ ID NO:78, SEQ IDNO:81, SEQ ID NO:84, SEQ ID NO:87, and SEQ ID NO:90.
 10. The assay ofclaim 8 wherein said first probe further comprises a first label andsaid second probe further comprises a second label.
 11. The assay ofclaim 10, wherein both labels are selected from a dye, a radioactiveisotope, a chemiluminescent label, and an enzyme.
 12. The assay of claim11, wherein the dye comprises a fluorescein dye, a rhodamine dye, or acyanine dye.
 13. The assay of claim 12, wherein the dye is a fluoresceindye.
 14. The assay of claim 13, wherein said first probe is labeled withFAM™ dye and said second probe is labeled with VIC® dye.
 15. The assayof claim 8, further comprising preparing the sample for PCRamplification prior to hybridizing.
 16. The assay of claim 15, whereinsaid preparing comprises at least one of the following processes: (1)bacterial enrichment, (2) separation of bacterial cells from the sample,(3) cell lysis, and (4) total DNA extraction.
 17. The assay of claim 8,wherein the sample comprises a food or a water sample.
 18. The method ofclaim 17, wherein the food sample comprises a selectively enriched foodmatrix.
 19. The assay of claim 8, wherein said amplifying is bypolymerase chain reaction.
 20. The assay of claim 8, wherein saidhybridizing and amplifying of said first pair of polynucleotide primersoccurs in a first vessel and said hybridizing and amplifying of saidsecond pair of polynucleotide primers occurs in a second vessel.
 21. Theassay of claim 8, wherein said hybridizing and amplifying of said firstpair of polynucleotide primers and said hybridizing and amplifying ofsaid second pair of polynucleotide primers occurs in a single vessel.22. The assay of claim 19, wherein said detection is a real-time assay.23. The assay of claim 22, wherein said real-time assay is a SYBR® Greendye assay.
 24. The assay of claim 19, wherein said detection is aTaqMan® assay. 25-74. (canceled)